Ischemia-reperfusion (I-R) in organs or anoxia-reoxygenation (A-R) in cells leads to cell death, oxidant stress, and organ dysfunction. Lung I-R is likely the inciting event leading to acute lung injury during lung transplantation/surgery, thromboembolectomy, pulmonary embolism, and re-expansion pulmonary edema, all of which lead to clinically significant respiratory failure but for which no specific therapies exist. Therefore, identifying protective mechanisms will be critical to the development of effective interventions. Heme oxygenase-1 (HO-1) is an important protective molecule but the underlying molecular mechanisms and responsible cell type(s) are poorly understood. HO-1 is the highly inducible isoform of heme oxygenase, the rate-limiting enzyme in heme degradation. Using lung-targeted HO-1 siRNA, we confirmed that endogenous HO-1 induction has important protective effects in lung endothelial cells and in vivo. Recently, we have found that endothelial STAT3 is critical to the protective effects of HO-1 during lethal oxidant injury and that an antioxidant molecule, heat shock protein, Hsp70, is modulated by STAT3. We have also generated endothelial-targeted HO-1 transgenic mice and HO-1 floxed mice, which will serve as valuable tools to explore the specific role of endothelial HO-1 in vivo. These observations have led us to propose the overall hypothesis that endothelial cell HO-1 mediates protection via endothelial STAT3-Hsp70-dependent anti-oxidant pathways during A-R/I-R injury. In order to test this hypothesis we will subject lung endothelial cells to A-R injury and mice to lung I-R injury in the following Specific Aims: 1) Determine the contribution of STAT3 to the anti-oxidant effects of HO-1 in lung endothelial cells and mouse lung, 2) Delineate the role of Hsp70 in mediating the protective effects of STAT3 and HO-1 in lung endothelial cells and mouse lung, and 3) Determine the specific contribution of endothelial-derived HO-1 in mediating protection in vivo. Upon completion of the studies, we will gain important insights into the role of the endothelium and the ways in which protective molecules such as HO-1 exert their effects during A-R/I-R injury and thereby identify novel therapeutic targets. PROJECT NARRATIVE. The overall goal of our project is to understand the ways in which the lung responds to and protects itself against injury. Respiratory failure after lung transplantation, the removal of life-threatening lung clots, and other major lung surgery is due to the transient cessation of blood flow followed by re-establishment of blood flow (ischemia-reperfusion) and carries a high mortality with limited options for intervention. We have identified novel mechanisms whereby a protein that we already possess, heme oxygenase-1, can protect against ischemia-reperfusion lung injury, and have also created powerful tools with which we can explore these mechanisms fully, in the hopes of applying our findings to the design of effective therapies against respiratory failure.